The present invention relates to hybrid toxin fragments, and toxins comprising them, derived from Bacillus thuringiensis insecticidal crystal proteins.
Bacillus thuringiensis (hereinafter B.t.) is capable of producing proteins that accumulate intra-cellularly as crystals. These crystal proteins are toxic to a number of insect larvae. Based on sequence homology and insecticidal specificity, crystal proteins have been categorized into different classes. Best studied are the CryI class of proteins, which are produced as 140 kDa protoxins and are active towards lepidopterans.
To some extent, the mode of action of crystal proteins has been elucidated. After oral uptake, the crystals dissolve in the alkaline environment of the larval midgul The solubilized proteins are subsequently processed by midgut proteinases to a proteinase-resistant toxic fragment of about 65 kDa, which binds to receptors on epithelial cells of the insect midgut and penetrates the cell membrane. This eventually leads to bursting of the cells and death of the larvae.
The activity spectrum of a particular crystal protein is to a large extent determined by the occurrence of receptors on the midgut epithelial cells of susceptible insects. The activity spectrum is co-determined by the efficiency of solubilization of the crystal protein and its proteolytic activation in vivo.
The importance of the binding of the crystal protein to midgut epithelial receptors is further demonstrated where insects have developed resistance to one of the crystal proteins, such that the binding of crystal proteins to midgut epithelial cells in resistant insects is significantly reduced.
Toxic fragments of crystal proteins are thought to be composed of three distinct structural domains. Domain I, the most N-terminal domain, consists of 7 xcex1-helices. Domain II comprises 3 xcex2-sheets. Domain III, the most C-terminal domain, folds into a xcex2-sandwich. If projected on CryI sequences, domain I runs from about amino acid residues 28 to 260, domain II from about 260 to 5 460, and domain m from about 460 to 600.
The present invention concerns hybrid crystal proteins particularly, though not exclusively, involving CryIC together with CryIE, CryIA, or CryIG. The nucleotide sequence of the CryIC gene from B.t. sub. sp. entomocidus 60.5 is given in SEQ ID NO:1, and the corresponding amino acid sequence of the protein encoded by said nucleotide sequence is given in SEQ ID NO:2. The Id nucleotide sequence of the CryIE gene from B.t. sub. sp. kenyae 4FI is given in SEQ ID No.3, and the corresponding amino acid sequence of the protein encoded by said nucleotide sequence is given in SEQ ID NO:4. The nucleotide sequence of a B.t. CryIG gene is given in SEQ ID NO:9, and the corresponding amino acid sequence of the protein encoded by said nucleotide sequence is given in SEQ ID NO:10. These proteins are toxic to lepidopterans, but within this order of insects, each protein has different specificity. CryIC, for example, is particularly active against S. exigua and M. brassicae. 
According to the present invention, there is provided an isolated B.t. hybrid toxin fragment comprising at its C-terminus domain III of a first Cry protein, or a part of said domain or a protein substantially similar to said domain; and comprising at its N-terminus the N-terminal region of a second Cry protein, or a part of said region or a protein substantially similar to said region. For example, a preferred B.t. hybrid toxin fragment according to the present invention comprises at its C-terminus domain III of a first Cry protein and comprises at its N-terminus domains I and II of a second Cry protein. A preferred fragment is one that does not bind to the CryIC binding site in an insect gut when it comprises at its C-terminus domain III of CryIC, or a part of said domain or a protein substantially similar to said domain; or one that does not bind to a CryIA binding site when it comprises at its C-terminus domain m of CryIA, or a part of said domain or a protein substantially similar to said domain.
In the context of the present invention, xe2x80x9csubstantially similarxe2x80x9d means a pure protein having an amino acid sequence that is at least 75% similar to the sequence of a protein according to the invention. It is preferred that the degree of similarity is at least 85%, more preferred that the degree of similarity is at least 90%, and still more preferred that the degree of similarity is at least 95%. In the context of the present invention, two amino acid sequences with at least 75%, 85%, 90%, or 95% similarity to each other have at least 75%, 85%, 90%, or 95% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 6 gaps, with the proviso that, with respect to the gaps, a total not more than 15 amino acid residues are affected. For the purpose of the present invention, conservative replacements may be made between amino acids within the following groups:
(i) Serine and Threonine;
(ii) Glutamic acid and Aspartic acid;
(iii) Arginine and Lysine;
(iv) Asparagine and Glutamine;
(v) Isoleucine, Leucine, Valine, and Methionine;
(vi) Phenylalanine, Tyrosine, and Tryptophan; and
(vii) Alanine and Glycine,
with the proviso that in SEQ ID NO:6, Ser and Tyr are conservative replacements at position 620, and Ala and Glu are conservative replacements at position 618; and that SEQ ID NO:8, Ser and Tyr are conservative replacements at position 627, and Ala and Glu are conservative replacements at position 625.
In the context of the present invention, xe2x80x9cpartxe2x80x9d of a protein means a peptide comprised by said protein and having at least 80% of the consecutive sequence thereof.
In the context of the present invention, xe2x80x9cbinding sitexe2x80x9d means a site on a molecule wherein the binding between site and toxin is reversible such that the Ka between site and toxin is in the order of at least 104dm3 molexe2x88x921.
The toxin fragment may comprise at its N-terminus the N-terminal region of any insecticidal protein from B.t. being commonly known as xe2x80x9cCryxe2x80x9d or xe2x80x9cCytxe2x80x9d, including: CryLA(a), CryIA(b) CryIA(c), CryIB, CryIC, CryID, CryIE, CryIF, CryIG, CryIH, CryIIA, CryIB, CryIIC, CryIIIA, CryIIIB, CryIIIB(b), CryIVA, CryrVB CryIVC, CryIVD, CYTA, CryX1(IIIC), CryX2(MD), CryX3, CryV, and CryX4, or a part of said region or a protein substantially similar to said region. The toxin fragment may comprise at its C-terminus domain m of CryIC, or a part of said domain or a protein substantially similar to said domain.
Thus, the fragment may comprise domain II of CryIE, CryIB, CryID, CryIA, or CryIG, or a part of said domain II or a protein substantially simidlar to said domain II, and domain m of CryIC or a part of said domain III or a protein substantially similar to said domain III. It is particularly preferred that the fragment comprises domains I and II of CryIE, CryIB, CryID, CryIA, or CryIG, or a part thereof or a protein substantially similar to said domains I and II, and domain III of CryIC or a part thereof or a protein substantially similar to said domain III.
It is most preferred that the toxin fragment comprises a region at its C-terminus comprising the sequence from amino acid position 454 to position 602 of CryIC, or a sequence substantially similar to said sequence. The fragment may comprise a region at its C-terminus comprising the sequence from amino acid position 478 to 602 of CryIC, or a sequence substantially similar to said sequence, with the proviso that if the sequence comprising amino acids 478 to 602 of CryIC is fused directly to the C-terminus of domain II of CryIA, CryIB, CryID, CryIE, or CryIG, then the folding of the fusion product is satisfactory to yield an insecticidal component of the fragment. The routineer in the art will recognize that it may be necessary to add a peptide region to the C-terminus of domain II that spaces the C-terminal region of CryIC apart, thus enabling it to fold in such a way as to exhibit insecticidal activity.
It is most particularly preferred that the toxin fragment according to the invention comprises one of the following:
i) an amino acid sequence from about amino acid 1 to about amino acid 620 in SEQ ID NO:6, or an amino acid sequence from about amino acid 1 to about amino acid 620 in SEQ ID NO:6, wherein with respect to said sequence, at least one of the following alterations is present:
Ile at position 609 is replaced with Leu,
Ala at position 618 is replaced with Glu,
Ser at position 620 is replaced with Tyr;
ii) an amino acid sequence from about amino acid 1 to about amino acid 627 in SEQ ID NO:8, or an amino acid sequence from about amino acid 1 to about amino acid 627 in SEQ ID NO:8, wherein with respect to said sequence, at least one of the following alterations is present:
Ile at position 616 is replaced with Leu,
Ala at position 625 is replaced with Glu,
Ser at position 627 is replaced with Tyr; and
iii) an amino acid sequence from about amino acid 1 to about amino acid 602 in SEQ ID NO:12.
Whatever amino acid alterations are permitted, however, one or more of the following residues indicated sequence-wise with respect to the CryIC sequence is invariable: Phe (501), Val (478), Trp (479), and Thr (486).
The invention also includes a hybrid toxin comprising the above disclosed fragment or a toxin at least 85% similar to such a hybrid toxin, which has substantially similar insecticidal activity or receptor binding properties.
The invention still further includes pure proteins that are at least 90% similar to the toxin fragments or hybrid toxins according to the invention.
The invention still further includes recombinant DNA comprising a sequence encoding a protein comprising an amino acid sequence of one of the above-disclosed toxins or fragments thereof. The invention still further includes recombinant DNA comprising the sequence from about nucleotide 1 to about nucleotide 1860 given in SEQ ID NO:5, or DNA similar thereto encoding a substantially similar protein; or recombinant DNA comprising the sequence from about nucleotide 1 to about nucleotide 1881 in SEQ ID NO:7, or DNA similar thereto encoding a substantially similar protein; or recombinant DNA comprising the sequence from about nucleotide 1 to about nucleotide 1806 in SEQ ID NO:11, or DNA similar thereto encoding a substantially similar protein.
In the context of the present invention, xe2x80x9csimilar DNAxe2x80x9d means a test sequence that is capable of hybridizing to the inventive recombinant sequence. When the test and inventive sequences are double stranded, the nucleic acid constituting the test sequence preferably has a TM within 20xc2x0 C. of that of the inventive sequence. In the case that the test and inventive sequences are mixed together and denatured simultaneously, the TM values of the sequences are preferably within 10xc2x0 C. of each other. More preferably, the hybridization is performed under stringent conditions, with either the test or inventive DNA preferably being supported. Thus, either a denatured test or inventive, sequence is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of between 50xc2x0 C. and 70xc2x0 C. in double strength citrate buffered saline containing 0.1% SDS, followed by rinsing of the support at the same temperature but with a buffer having a reduced SC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, such reduced concentration buffers are typically single strength SC containing 0.1% SDS, half strength SC containing 0.1% SDS and one tenth strength SC containing in 0.1% SDS. Sequences having the greatest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the test and inventive sequences are so similar that the hybridization between them is substantially unaffected by washing or incubation in one tenth strength sodium citrate buffer containing0.1% SDS. Typical stringent conditions are as follows: hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 pH 7.0, 1 mM EDTA at 50xc2x0 C.; wash with 2xc3x97SSC, 1% SDS, at 50xc2x0 C.
The recombinant DNA may further encode a protein having herbicide resistance, plant growth-promoting, anti-fungal, anti bacterial, anti-viral, and/or anti-nematode properties. In the case that the DNA is to be introduced into a heterologous organism, it may be modified to remove known mRNA instability motifs (such as AT rich regions) and polyadenylation signals, and/or codons that are preferred by the organism into which the recombinant DNA is to be inserted may be used so that expression of the thus modified DNA in the organism yields substantially similar protein to that obtained by expression of the unmodified recombinant DNA in the organism in which the protein components of the hybrid toxin or toxin fragments are endogenous.
The invention still further includes a DNA sequence complementary to one that hybridizes under stringent conditions with the recombinant DNA according to the invention.
Also included in the present invention are the following: a vector containing such a recombinant (or complementary thereto) DNA: sequence; a plant or microorganism that includes and enables expression of such DNA; plants transformed with such DNA; the progeny of such plants that contain the DNA stably incorporated and hereditable in a Mendelian manner, and/or the, seeds of such plants and such progeny.
The invention still further includes protein derived from expression of the recombinant DNA of the invention, and insecticidal protein produced by expression of the recombinant DNA within plants transformed therewith.
The invention still further includes the following: an insecticidal composition containing one or more of the toxin fragments or toxins comprising them according to the invention; a process for combating insects that comprises exposing them to such fragments or toxins or compositions; and an extraction process for obtaining insecticidal proteins from organic material containing them, comprising submitting the material to maceration and solvent extraction.